Direct Observation of RecA-Mediated Four-strand Exchangeat the Single-Molecule Level

• Main Authors: Mu-Ni Hsu, 徐慕霓
• Other Authors: 李弘文
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/38620454989848055371

碩士 === 國立臺灣大學 === 化學研究所 === 98 === E. coli RecA protein is required for repairing damaged DNA through homologous recombination pathway. The assembled RecA/DNA nucleoprotein filaments catalyze the reactions that paired and exchange with homologous DNA sequence. We used single-molecule tethered particle motion (TPM) experiments to directly observe the strand exchange process catalyzed by RecA in real-time. The surface-bound DNA is a duplex DNA with a bead could be displaced due to four-strand exchange. Calculate the percentage of leaving tethers under different condition to obtain the properties of four-strand exchange. The RecA-mediated strand exchange efficiency is higher for ATP than ATPγS, suggesting that ATP hydrolysis by RecA is required to complete strand exchange. In addition, the RecA-mediate strand exchange is shown to progress either from 5’end or from 3’end in our experiment. Furthermore quantitative analysis of DNA exchanging time courses using ‘‘n-step’’ sequential mechanisms, can reveal information about the rate constant of four-strand exchange reaction and the average number of basepairs in the strand exchange cycle. The result indicates that the rate is larger for longer single-stranded gap and 5’overhang.